The long-term objective of this project is to understand the catalytic mechanism of acetyl-CoA synthase/carbon monoxide dehydrogenase (ACS/CODH) from Moorella thermoacetica. We have five specific aims for the next four years. First, we have recently discovered that the catalytically active form of isolated alpha is a dimer not a monomer, with 1 catalytically active subunit and 1 structural subunit. We will characterize the dimer, including by X-ray diffraction, and determine whether subunit asymmetry can be eliminated. Second, we will use NMR and X-ray diffraction to determine whether methyl and acetyl catalytic intermediates bind to the proximal Ni (Nip) of the A-cluster active site. If so, Nip would be only the third metalloprotein center known to support such unusual organometallic bonds. Third, we will use biophysics and redox chemistry to determine whether Nip achieves a zero-valent oxidation state during catalysis. If so, this would be unprecedented in biology. Fourth, the alpha subunit can exist in 2 conformations and undergo a conformational change during each catalytic cycle. We will develop a fluorescence (FRET) assay to sense these changes and determine which conformation is present at each catalytic step. Finally, we will expand our recently developed kinetic/mathematical model of acetyl-CoA synthesis to include additional mechanistic features, such as the conformational changes, pH effects, and cooperative CO inhibition. Such models represent the most rigorous means of testing enzyme mechanisms. Relavance to Public Health: Clostridium difficile, which contains this enzyme ACS/CODH, causes antibiotic-associated colitis, toxic megacolon, intestinal perforations and even death in humans. Spores of Clostridium novyi-NT, which almost certainly contain ACS/CODH, injected into cancerous tumors of mice grow exclusively in the anaerobic environment of the tumor, killing cancerous cells and increasing survival rates. Our mechanistic study of ACS/CODH will help define the metabolic roles played by the same enzymes in these pathogens, and identify strategies for preventing the proliferation of C. difficile in intestines and for encouraging the proliferation of C. novyi in cancerous cells. Also, ACS/CODH removes CO from the atmosphere and degrades TNT from abandoned military sites. ACS/CODH is involved in C1 metabolism and it contains a tunnel through which CO migrates, impacting the field of metabolic channeling.